Infectious disease is the second leading cause of death worldwide, the growing number of antibiotic-resistant microbes threatens to further worsen this public crisis, but the pipeline for new antibiotics is running dry. Diabetes affects nearly 26 million people in the US, as many as 1 in 3 American adults will have diabetes in 2050 if present trends continue, and yet current approved therapies suffer from inadequate efficacy and/or liabilities. The discovery of platensimycin (PTM) and platencin (PTN) as selective and potent bacterial and mammalian fatty acid synthase inhibitors and the demonstrated efficacy of PTM and PTN in animal models as a new class of antibacterial and antidiabetic drug leads are therefore of exceptional importance. However, development of PTM and PTN into commercial drugs faces formidable obstacles, including improvement of the drugs' pharmacokinetics and the inevitable emergence of resistance. We propose in this application to: (i) characterize the PTM and PTN biosynthetic pathways, (ii) engineer the PTM and PTN biosynthetic machinery to generate novel analogues, and (iii) investigate PTM and PTN self-resistance in the native producers and acquired resistance in clinically relevant pathogens. Our hypotheses are: (i) studies of PTM and PTN biosynthesis promise to uncover new chemistry and enzymology, (ii) exploration of diterpenoid synthase specificity and tailoring enzyme promiscuity provides an outstanding opportunity to generate novel analogues, (iii) comparison and contrast of PTM and PTN self-resistance in the producers to acquired resistance in pathogens provides an excellent opportunity to understand, and therefore predict and combat future PTM or PTN resistance in clinical settings. The long-term goal of our research is to develop PTM and PTN into a new class of clinically useful antibacterial and antidiabetic drugs. The specific aims for this grant period are: (i) functional characterization of the PTM and PTN biosynthetic machinery in vivo and biochemical characterization of the novel enzymes for PTM and PTN biosynthesis in vitro, (ii) engineering of the PTM and PTN biosynthetic machinery for novel PTM and PTN analogues, (iii) investigation of the molecular mechanisms of PTM and PTN self-resistance in the producers and acquired resistance in pathogens. The outcomes of the proposed studies will include: (i) PTM and PTN biosynthesis as a model system for bacterial diterpenoid natural products, (ii) novel chemistry and enzymology for diterpenoid biosynthesis, (iii) PTM and PTN analogues with improved pharmacokinetic properties as antibacterial and antidiabetic drug leads, and (iv) molecular mechanisms of PTM and PTN resistance.